Aspartic proteases from Silybum marianum: different plant-specific inserts, different destinations.

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conclusionPlant-specific inserts (PSIs) are supposed to direct typical aspartic peptidase (AP) to the vacuole. Two typical AP precursors possess distinct PSIs. One PSI directs the peptidase to the vacuole, while the other cannot. Typical plant aspartic peptidases (APs) are proteolytic enzymes unique to plants. What distinguishes this group of peptidases is the saposin-like domain known as the plant-specific insert (PSI), which is present in all typical AP zymogens. In this study, we cloned and characterized two novel typical APs from Silybum marianum flowers, designated AP-Sm1 and AP-Sm2. Using in silico analysis and phylogenetic comparisons, we elucidated the structural features of their zymogens, including conserved motifs and catalytic subsites. Our findings suggest that these enzymes originated from the duplication of an ancestral AP gene. Although AP-Sm1 and AP-Sm2 share sequence and structural similarities with other plant APs, they have potential differences in substrate specificity, which may be attributed to variations in the S3, S1', and S3' catalytic subsites. We also identified distinct putative N-glycosylation patterns between the two enzymes, with AP-Sm1 being glycosylated within its PSI domain. This domain has been suggested as a player in environmental adaptation and may influence the trafficking of typical AP zymogens through the secretory pathway. We observed differences in the subcellular localization of mRFP-fused AP-Sm1 and AP-Sm2 when the C-terminal vacuolar sorting determinant (ctVSD) was non-functional. While AP-Sm2 localized to the vacuole, AP-Sm1 was detected in the apoplast. As suggested by other authors, the differential glycosylation profile within PSI domains might modulate intracellular trafficking, potentially contributing to its distinct localization pattern. These findings highlight the potential of AP PSIs as valuable models for further studies on protein trafficking mechanisms in plant cells.